Fine Structure of Negatively Stained Plant Mitochondria

Recently investigators have reported knob-like particles on mitochondrial membranes isolated from different organisms (1, 2, 4, 5). This report presents observations on the structure of plant mitochondria, negatively stained with phospho-tungstic acid, which show knob-like projections on the membranes. MATERIALS AND METHODS Castor beans (Ricinus communis) were soaked for 24 hours in distilled water and sown in moist vermicu-lite. The seeds were allowed to germinate for 41/~ days in the dark at 29°C. At the end of this period, mitochondria were isolated from endosperms according to Wiskich and Bonner (6), except that the final suspension was made in de-ionized water. Geronimo and Tanner (personal communication) of this department have obtained high enzymatic activity for endosperm mitochondria isolated from castor beans in the same manner. A small drop of the mitoehondrial fraction in water was placed on an electron microscope grid with a supporting film of carbon. A filter paper was used to remove the excess water until only a thin film remained on the grid. A drop of 2 per cent phospho-tungstic acid in water, adjusted to pH 6.5 with 10 per cent potassium hydroxide, was then added to this film and the excess removed as above. The grid was allowed to dry at room temperature. The electron micrographs were made in a Hitachi HU 11-A microscope using double condenser, and the photographs were taken at a screen magnification of 25,000. Structural details of mitochondrial membranes and cristae were distinguishable at an optical magnification of 140,000. RESULTS Examination of the negatively stained mito-chondrial fraction in the electron microscope showed intact mitochondria as wall as membrane fragments of varying size. Usually, the surfaces of membranes in intact and disrupted mitochondria showed lollipop-like particles (Fig. 1). These particles were more pronounced on the irregular ribbon like structures in the disrupted mitochondria than in the intact ones. No particles were seen on the outer membrane of the mimchondria. The particles seem to be composed of roughly spherical heads attached to the mitochondrial membrane by narrow stalks. The diameter of the heads falls within the 75-95 A range, and the length of the stalks, which are only occasionally clearly distinguishable (Fig. 2), within the 40-60 A range. The particles seem to be distributed at the rate of 100 per micron length of membrane surface. Occasionally, areas of the membrane appeared to be lacking the particles.